28 FIXATION 



size enable us to judge the quality of fixation at the level of resolu- 

 tion of the light-microscope. Thus, an object of microscopic size, 

 known to be smooth in outline in life, may appear irregular in an 

 electron micrograph, and the irregularities may be of such a size 

 that they would have been visible with the light-microscope in 

 life, had they existed. We have here clear evidence of faulty fixa- 

 tion for electron microscopy; and if the fixation is faulty at the 

 microscopical level, we cannot trust it at the submicroscopical. 

 So far as submicroscopical objects are concerned, we have no 

 direct means of knowing whether a fixative is reliable or not. It is 

 customary to rely upon a fixative that is known to give faithful 

 stabilization of form at the microscopical level. It would be safest 

 not to rely on the submicroscopical detail shown in electron 

 micrographs unless similar pictures are given after fixation by 

 several different fixatives, all known to be reliable at the micro- 

 scopical level. 



Many cytologists have examined separate living cells with the 

 light-microscope and noticed the changes produced in them when 

 a fixative was run below the coverslip. The classical work of this 

 kind was done by Strangeways and Canti (1927) with cultured 

 cells, by dark-ground microscopy. The invention of the phase- 

 contrast microscope led to a whole series of similar studies. Such 

 work is of great interest and value, but it is of course an incom- 

 plete way of studying fixation, for only the primary artifacts are 

 usually studied, and no evidence is obtained about the ability of the 

 fixative to stabilize the form of the cellular components against 

 subsequent treatment. In fact, fixatives are not tested as such in 

 these studies, but only as preservatives. 



A defect of the method described in the last paragraph is that 

 the cells studied are immediately exposed to the action of the 

 fixative. Now when a piece of tissue is fixed in the ordinary way, 

 the cells that are thus exposed and not protected by any special 

 membrane (such as the free border of the intestinal epithelium of 

 vertebrates) are often seriously distorted, while those below, 

 protected as they are by overlying cells or membranes, are well 

 fixed. Even in a piece of tissue only a millimetre or so in diameter, 

 the great majority of the cells are not superficial. It therefore 

 follows that one might reject a fixative wrongly on the basis of 

 observations made on separate cells. 



It is realistic to test fixatives on small pieces of tissue that will be 

 embedded and sectioned, with subsequent dyeing and mounting 



